The purpose of this proposal is to study the mechanism of replication of the bacterial chromosome, especially in relation to the possible involvement of RNA primers in discontinuous DNA synthesis. The role of RNA polymerase in the synthesis of such primers will be studied in Bacillus brevis, which seems especially suitable for this kind of investigation for two reasons. Firstly, preliminary experiments have shown that DNA synthesis in B. brevis is inhibited by rifampicin and streptolydigin, suggesting a dual function of RNA polymerase in transcription and replication. Secondly, I have developed an in vitro permeable cell system in which DNA synthesis is dependent on the simultaneous function of RNA polymerase. This system will be expolited to define the role of RNA synthesis in DNA replication. New methods are proposed for the isolation and characterization of RNA primers attached to growing DNA chains. This involves the use of 5-mercuri-dCTP for the isolation of nascent DNA chains on the thiol-Sepharose columns, together with 2',3'-dideoxy TTP to inhibit nick translation and the concomitant degradation of RNA primers. These methods avoid the disadvantages inherent in the procedures used by others in attempts to isolate DNA-linked RNA primers.